Amish Nemaline Myopathy (ANM) is an autosomal recessive muscle disorder found among the Old Order Amish in Pennsylvania and Ohio, affecting 1 out of every ~500 births. The ANM allele contains a nonsense mutation in the slow skeletal muscle troponin T (TnT) gene (TNNT1), which results in truncation of the TnT protein at amino acid 179. This truncated slow TnT cannot incorporate into myofibrils and is degraded inside the myocyte. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. The affected infants are clinically normal at birth but rapidly develop the ANM symptoms that usually result in death from respiratory failure during the second year. No effective treatment is available. This research project aims to understanding the pathogenesis of ANM and the pathophysiology of ANM muscle for the optimal goal of developing a cure of the disease. The following three specific aims are proposed: Aim I: To characterize animal models deficient in slow TnT. We shall study the skeletal muscle function in a slow TnT knockdown mouse model in which lowered slow TnT gene expression results in decreased slow TnT protein, muscle atrophy and a switch to fast fiber phenotypes with decreased tolerance to fatigue. We shall also study the phenotype of a mouse model in which the ANM nonsense mutation is knocked in the slow TnT gene to investigate the pathogenesis and disease progression. Aim II: To examine the conditional effects of slow TnT gene haploidy and cytotoxicity of the truncated slow TnT on ANM pathogenesis and pathophysiology. ANM heterozygotes have reported circumstantial muscle symptoms. We shall investigate the potential effect of half dosage of slow TnT gene on muscle function in heterozygote slow TnT knock-in mutant mice. Non-myofilament-associated TnT fragments exhibit cytotoxicity. We shall examine whether and how this potential cytotoxicity of the ANM slow TnT fragment contributes to muscle degeneration in ANM patients. Aim III: To study the regulation and function of cardiac TnT in skeletal muscle in order to develop therapeutic compensation in ANM patients. We have found evidence in a subtype of ANM that the loss of slow skeletal TnT function in ANM may be partially compensated for by the continuing expression of cardiac TnT in skeletal muscles. Functional significance and activation of this compensatory cardiac TnT gene expression will be investigated at protein, muscle cell and tissue, and animal levels toward the development of a specific treatment for ANM. In an alternative approach, suppression of the ANM nonsense stop codon will also be explored using emerging reagents. Based on progresses we have made to date, these studies employ state-of-the-art molecular genetic, biochemical, cell biological and physiological methods and novel experimental systems. The results will significantly further the pursuit of an effective therapy for this devastating disease. PUBLIC HEALTH RELEVANCE: Nemaline myopathies are a group of neuromuscular disorders characterized by muscle weakness and rod-shaped "nemaline" inclusions in skeletal muscle fibers. All known inherited nemaline myopathies are caused by mutations in sarcomeric thin filament proteins. Amish Nemaline Myopathy (ANM) is a lethal inherited nemaline myopathy present at very high incidences (1 out of 500 births) in the Old Order Amish communities in Pennsylvania and Ohio. The genetic basis of ANM is a single nucleotide nonsense mutation in the gene encoding the slow skeletal muscle isoform of troponin T (TnT), a muscle-specific Ca2+- regulatory protein. This nonsense mutation truncates the slow TnT polypeptide at amino acid 179 and renders the TnT protein incapable of incorporating into myofibrils and being rapidly degraded. This loss of function mechanism is consistent with the autosomal recessive inheritance of the disease. Phenotypically, individuals with ANM suffer from muscle tremors, contractures and hypotonia. Symptoms are trivial at birth but rapidly worsen, usually resulting in death from respiratory failure during the second year. No effective treatment is currently available. Although ANM is a rare disease in the general population, its uniformly devastating progression combined with an already elucidated molecular cause merits much further investigation. Two to three ANM babies are born every year in Pennsylvania and Ohio;none will live to be 3 years old. We are currently the only research team in the world working on ANM and our progress thus far has already given the affected families and the general Amish community a hope for effective treatments in the near future. This research project aims to further our progress towards the development of a therapy by investigating TnT isoform function and regulation. We will study slow TnT-deficient animal models for the understanding of ANM pathology and muscle function. We will examine the potential effect of decreased slow TnT gene dosage on muscle function and the cytotoxicity of truncated slow TnT for links to muscle degeneration in ANM. Together with exploring the possibility of suppressing the ANM nonsense stop codon, we will focus on investigating the functional significance and activation of compensatory cardiac TnT expression in slow TnT-deficient skeletal muscle for use as a specific treatment for ANM. ANM is the only known disease caused by a recessive mutation in a TnT gene. By investigating the pathophysiology of ANM, we will also gain important insights into the functional significance of different TnT isoforms and the Ca2+-regulation of striated muscle contraction, a fundamental topic in biology and medicine.